A Combinatorial Synthetic Strategy for Developing Genome-Editing Protein-Delivery Agents Targeting Mouse Retina

CRISPR Cas9 based gene editing technologies offer promise for treating inherited retinal diseases. However, safe and efficient ocular delivery of precision editors remains challenging. To address this challenge, we designed a new class of Coomassie brilliant blue CBB derived lipidoids that bind and deliver ribonucleoprotein RNP complexes. These CBB lipidoids leverage the dyes intrinsic affinity for proteins and facilitate cytosolic delivery. First, use of a focused library of CBB lipidoids enabled efficient in vitro delivery of Cre recombinase. Subsequently, subretinal injection of these lipidoids into mTmG mice achieved robust Cre mediated recombination in the retinal pigment epithelium and photoreceptors. We then employed the CBB lipidoid platform to deliver adenine base editor ABE-ribonucleoproteins RNP both in vitro and in vivo. Incorporating CBB lipidoids into liposomes containing ionizable lipids further improved the colloidal stability and delivery efficiency of ABE RNP complexes. Among the tested molecules, CBB11 stood out for facilitating precise in vitro and in vivo ABE mediated gene editing. Delivery of liposome CBB11 RNP complexes resulted in a 120 fold increase in precise base editing compared to RNP alone and restored the scotopic ERG b wave response in the rd12 mouse model of retinal degeneration. We further report acute structural and functional tolerability in wild type mouse retina. These results demonstrate the potential of CBB augmented, transiently delivered liposome RNP systems for therapeutic gene editing in the eye, paving the way for single dose precision medicines to treat inherited retinal diseases IRDs.